Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A

Abstract In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits―Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS–tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

Crystal structures of KEOPS subcomplexes allowed reconstruction of structural models for KEOPS from archaea ( 3 ,44 ), yeast ( 56 ) and humans ( 18 ,57 ).These three models of KEOPS showed that the core subunits adopt a conserved linear architecture depicted as Pcc1 / LAGE3-Kae1 / OSGEP-Bud32 / PRPK-Cgi121 / TPRKB ( 31 ,32 ).Gon7 / GON7 is an intrinsically disordered protein and interacts solely with Pcc1 / LA GE3 in KEOPS ( 18 , 56 ).Recently, a paralog of archaean Pcc1, dubbed Pcc2, interacts with Pcc1 in a manner that is analogous to Gon7 / GON7 ( 46 ).Biochemical analysis demonstrated that the four-subunit KEOPS from archaea forms a dimer ( 20 ,44 ) whereas the five-subunit KEOPSs from yeast and humans cannot form a dimer due to the presence of Gon7 / GON7 ( 12 , 18 , 56 ).The dimerization of Pcc1 leads to formation of archaean KEOPS dimer that is required for in vitro tRNA t 6 A biosynthesis ( 44 ,46 ).Subtraction of Pcc1 from archaean KEOPS leads to dead t 6 A-catalytic activity of the subcomplex Kae1-Bud32-Cgi121 ( 20 ).Deletion of Gon7 / GON7 severely affects tRNA t 6 A biosynthesis by KEOPS in yeast and humans ( 12 , 18 , 34 ).Likewise, replacement of Pcc1 by Pcc2 in archaean KEOPS still sustains the dimeric state but leads to loss of t 6 A catalytic activity ( 46 ).Crystal structure of M. jannaschii ( Mj ) Cgi121-tRNA complex allowed generation of a structural model of archaean KEOPS-tRNA complex ( 45 ).According to this model, anticodon stem loop of tRNA is anchored in the concave formed between Pcc1 and Kae1, allowing tRNA A37 to protrude into the t 6 A-catalytic center of Kae1; D stem loop of tRNA simultaneously makes contacts with Kae1 and Bud32; 3 CCA end is bound by Cgi121; T ψ C stem loop does not participate in direct interaction with KEOPS.This model delineates a plausible binding mode for KEOPS-tRNA and explains the roles of individual subunits ( 31 ,45 ).However, precise molecular interactions between KEOPS and tRNA are still not determined, e.g. the binding of tRNA A37 in catalytic site of Kae1 is still enigmatic.Nonetheless, a manual adjustment of tRNA crystal structure is needed to geometrically fit an extended surface of the four subunits of KEOPS ( 45 ), suggesting that binding of tRNA to KEOPS might mutually induce large conformational changes in structures of KEOPS and tRNA.Structural analysis and biochemical validations demonstrated that t 6 A-catalytic activity of KEOPS necessitates an ATP to ADP hydrolysis by Bud32 ( 19 , 20 , 45 , 56 , 57 ).At present, it's poorly understood as how KEOPS subunits cooperate to regulate KEOPS-tRNA assembly and t 6 A modification efficiency.Therefore, an atomic structure of a complete KEOPS complex is desirable to investigate the precise roles and molecular workings of KEOPS in tRNA t 6 A biosynthesis.
Comparative genomics analysis revealed that orthologs of the four core subunits of KEOPS-Kae1, Bud32, Cgi121 and Pcc1-are encoded in Arabidopsis thaliana genome ( 11 ) .However, the biochemical functions and structures of A. thaliana KEOPS proteins have not been characterized.Moreover, liquid chromatography-mass spectrometry (LC-MS) analysis revealed t 6 A and its hypermodified derivative-N 6methyl t 6 A (m 6 t 6 A) in tRNAs isolated from Arabidopsis thaliana ( 58 ).Yet, the biochemical pathway of tRNA t 6 A biosynthesis remains uncharacterized.In this study, we reconstituted an enzymatic biosynthesis of tRNA t 6 A using purified recombinant proteins of A. thaliana KEOPS (KAE1, BUD32, CGI121 and PCC1) and YRDC.We reconstituted A. thaliana KEOPS complex and investigated molecular mechanisms of A. thaliana KEOPS in tRNA t 6 A biosynthesis.Here, we report the cryo-EM structure of A. thaliana KEOPS and structurefunction relationship analysis of A. thaliana KEOPS-tRNA assembly, which extend our current mechanistic understandings of the ancient KEOPS machineries.
At KEOPS complex was reconstituted using purified KAE1-PCC1 and BUD32-CGI121, which were mixed at a molar ratio of 1:2 and applied to SEC for isolation of complete KEOPS complex.At KEOPS variants were prepared in same way using corresponding variants of KAE1-PCC1 or BUD32-CGI121.The oligomeric state of At KEOPS complex, subcomplexes and variants were determined by SEC (Superdex 200 Increase 10 / 300 GL column, HiLoad 16 / 600 Superdex 200 column, GE Healthcare) with reference to Sc KEOPS, of which the oligomeric state and molecular weight were determined by SAXS ( 56 ).Standard calibration and reference to other welldetermined proteins were used for comparing elution volumes and estimating the molecular weight.

Bulk tRNA extraction
Arabidopsis thaliana RNAs were extracted with TRIzol (Ther-moFisher Scientific) from seedlings grown for 3 days at 25 • C on Murashige and Skoog medium supplemented with 0.8% agar and 1% sucrose, followed by precipitation using absolute ethanol at -80 • C. Total RNAs were applied to 8 M urea-polyacrylamide gel (12%) electrophoresis (Urea-PAGE).Gel slices containing full-length tRNAs were cut out, followed by elution in buffer containing 500 mM NaAc pH 5.2 and precipitation in ethanol at -80 • C. The tRNA pellets were dissolved in buffer containing 50 mM Tris-HCl pH 8.0, 5 mM MgCl 2 and 100 mM KCl. Refolding of tRNAs was performed by heating up to 95 • C and gradient annealing at a rate of -1 • C / min.Yeast total RNAs were extracted with TRIzol from S. cerevisiae sua5 strain cells that were grown in YPD medium at 28 • C for 72 h ( 4 ).Bulk Sc tRNAs were purified following same protocols of separation, precipitation and refolding as for bulk At tRNAs.Concentration of tRNAs was determined by NanoDrop2000 (ThermoFisher Scientific).

In vitro transcription of tRNA
DNA templates of tRNA Arg CCU (Gene ID: 3771562), tRNA Arg UCU (Gene ID: 3767807), tRNA Thr CGU (Gene ID: 3768453), tRNA Ile AAU (Gene ID: 3770537), tRNA Lys UUU (Gene ID: 3768774), tRNA Ser GCU (Gene ID: 3769744), tRNA Met CAU (Gene ID: 3766659), tRNA Asn GUU (Gene ID: 3768376) and tRNA Ile UAU (Gene ID: 3768844) from Arabidopsis thaliana were prepared by overlap extension PCR using chemically synthesized primers, in which the forward primer contains a T7 promoter sequence at the 5 terminus.tRNA genes and primers are summarized in ( Supplementary Table S2 ).Run-off transcription was carried out using T7 RNA polymerase at 30 • C for 8 hours in a reaction mixture containing 40 mM Tris-HCl pH 8.0, 5 mM NTP mix, 5 mM DTT, 1 mM spermidine, 0.5% Triton X-100, 33 mM MgCl 2 , 3 μM T7 RNA polymerase, 10 μM pyrophosphatase and 15 mM GMP.RNA transcripts from in vitro transcription (IVT) were further purified by Urea-PAGE following protocols as for bulk tRNAs of Arabidopsis thaliana .The correct folding of IVT At tRNA was confirmed by Circular Dichroism spectra analysis ( 59 ).Concentrations of IVT tRNAs were determined by NanoDrop2000.

Enzymatic synthesis of tRNA t 6 A and LC-MS analysis of t 6 A nucleoside
For TC-AMP formation, 2 μM At YRDC was incubated with 4 mM L -threonine, 20 mM NaHCO 3 , 2 mM ATP and 5 mM MgCl 2 in a reaction buffer containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 1 mM TCEP for 10 minutes at 25 • C. For tRNA t 6 A formation, 2 μM At KEOPS and 20 μM tRNA were added to the TC-AMP biosynthesis reaction system and the mixture was incubated for 90 minutes at 30 • C followed by purification using 12% Urea-PAGE as described above.The dissolved t 6 A-tRNA was digested into single nucleoside using Nuclease P1 (0.1 U / ml, Sigma) and Alkaline Phosphatase (0.1 U / ml, Sigma).50 μl sample of the mononucleosides was chromatographed using a C18 column (5 μm, 4.6 × 250 mm, Agilent) at a flow rate of 0.8 ml / min using a mobile phase composed of 0.1% trifluoroacetic acid aqueous and methanol.The nucleosides were isocratically eluted with 5% methanol for 5 min and gradiently eluted with 5-40% methanol for 15 min and 40-98% methanol for 5 min.Nucleosides were identified by the retention time at 254 nm and the mass spectrometry detection (Agilent 6125B).Data collection and analysis were performed using the OpenLab software v3.5 (Agilent).Based on the integrated peak areas of A, U, C, G and t 6 A, the t 6 A modification efficiency was obtained by dividing the peak area ratio (t 6 A / A) by the number ( N ) ratio of 1 / ( N A -1). Three independent replicates were performed for all assays and data graphs were generated using GraphPad Prism.Error bars in quantification data represent standard deviations for triplicate measurements.
Cryo-EM sample preparation, data collection and processing, model building and validation 800 μg / ml At KEOPS was mixed with equal volume of tRNA Arg CCU at a molar ratio of 1:2.A drop of 3 μl sample was applied to a freshly glow-discharged Quantifoil gold grid (300 mesh, R0.6 / 1) and blotted for 2 s at 10 • C under 100% humidity in a FEI Vitrobot Mark IV (ThermoFisher Scientific).The grids were stored in the liquid nitrogen until data acquisition.Cryo-EM data were collected on the Titan Krios G3i microscope operated at 300 kV (ThermoFisher Scientific) and equipped with a BioQuantum K3 Imaging Filter direct electron detector (Gatan).The energy filter was used in zero-loss mode with a slit width of 20 eV.All the movies were automatically recorded with EPU software (ThermoFisher Scientific) at a nominal magnification of 105 000 x in super-resolution mode with a pixel size of 0.43 Å.The defocus range was from -0.9 to -2.7 μm.All the movie stacks were collected from three cryo-EM sessions.The total electron dose for each movie stack was 52-64 e − / Å 2 fractionated into 40 fractions over 3.2 s.
The entire image processing was carried out with cryoSPARC v3.3 ( 60 ).All the movie stacks were subjected to patch motion correction and patch CTF estimation.A total of 14 622 micrographs were selected to do the following procedure.Firstly, 1 470 particles were manually picked from 100 micrographs with varied defocus values and subjected to 2D classification.Good particles corresponding to those 2D class average maps showing clear density distribution were used for topaz training.In total, 736 244 picks were extracted with a box size of 512 pixels.After several rounds of 2D classification, 327 681 picks were retained and subjected to ab initio reconstruction into three models.Heterogeneous refinement was carried out to further clean the data set.A total of 232 232 picks from two classes were combined to perform the homogeneous refinement, yielding a resolution of 3.95 Å.After that, a further round of non-uniform refinement was performed to improve the resolution to 3.65 Å.An overall mask was generated and used in the local refinement, which finally improved the resolution to 3.2 Å.The resolution for all reconstructions were evaluated by using the gold standard Fourier Shell Correlation (FSC) of 0.143.Data collection and reconstruction parameters are presented in Supplementary Table S3 .

ATPase assay
The NADH-coupled ATPase assay was employed to analyze the hydrolysis of ATP to ADP ( 39 ,47 ).200 μl reaction mixture was made of 4 mM phosphoenolpyruvate, 0.5 mM NADH, 6 U / ml pyruvate kinase, 9 U / ml lactate dehydrogenase, 2 mM ATP and 5 μM proteins in the presence or absence of 10 μM tRNA Arg CCU in buffer containing 50 mM Tris -HCl pH 7.5, 100 mM NaCl, 50 mM KCl, 5 mM MgCl 2 and 1 mM DTT.The absorbance at 340 nm was recorded at an interval of 30 seconds for a total of 60 min at 25 • C with MULTISKAN GO microplate reader (ThermoFisher Scientific) using a 96-well plate.When ATP is hydrolyzed to ADP, NADH is gradually consumed, as reflected by the decrease in absorbance values.A standard curve of ADP against NADH absorbance was obtained to quantify the hydrolysis rate of ATP to ADP.For kinetic analysis, 2 μM protein and 0-50 μM ATP were used and kinetic parameters were calculated according to Michaelis-Menten equation.All the measurements were performed inde-pendently in triplicates.Error bars in the data represent standard deviations.

Electrophoretic mobility shift assay
For the native gel analysis, 20 μM At KEOPS was incubated with 20 μM IVT At tRNAs in the buffer containing 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM βmercaptoethanol and 20% (v / v) glycerol.The mixture was loaded onto a non-denaturating gel (2% agarose, 50 mM Tris-base pH 8.0 and 100 mM glycine) and electrophoresis ran for 1 hour at 100 V at 4 • C in pre-chilled buffer containing 50 mM Tris -base pH 8.0 and 100 mM glycine.tR-NAs were visualized under UV light at 254 nm after staining with Ethidium Bromide, and proteins were stained with Comassie Brilliant Blue. 5 6-FAM (6-Carboxyfluorescein)labelled At tRNA Arg CCU (5 -6FAM-tRNA Arg CCU ) was chemically synthesized (Tsingke) and was re-folded before use.0.1 μM 5 -6FAM-tRNA Arg CCU was incubated with At KEOPS proteins (0-0.5 μM) for gel-shift assay on a non-denaturing gel (1.6% agarose, 50 mM Tris-base pH 8.5 and 100 mM glycine).The electrophoresis ran for 1 hour at 100 V at 4 • C in pre-chilled buffer containing 50 mM Tris -base pH 8.5 and 100 mM glycine.The presence of 5 -6FAM-tRNA Arg CCU on gel was visualized at a wavelength of 488 nm by Pharos FX (Bio-Rad).10-fold amount of the original input protein in each lane was analyzed and visualized on a separate SDS-PAGE.

Microscale thermophoresis (MST)
50 nM 5 -6FAM-At tRNA Arg CCU was incubated with At KEOPS complex, subcomplexes or variants at increasing concentrations (0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 μM) in MST buffer (20 mM PBS pH 7.5, 300 mM NaCl and 0.05% (v / v) Tween 20).Measurements were performed at 25 • C in capillaries (MO-K022, NanoTemper Technologies) on the Monolith NT.115 (NanoTemper Technologies) using 20% LED and medium IR-laser power.Binding data was analyzed using MO.affinity analysis software (NanoTemper Technologies) and equilibrium dissociation constant ( K d ) values were fitted by the K d equation and model.All experiments were reproduced at least for three times using proteins from different batches.Error bars in data analysis represent standard deviations for triplicate measurements.

LC-MS / MS proteomic analysis
In the pull-down experiment, 0.5 g Arabidopsis thaliana seedlings were grounded in liquid nitrogen.Total proteins were recovered in 200 μl Lysis buffer supplemented with 1 mM PMSF, followed by centrifugation at 4 • C. The supernatant was incubated with 100 μg purified At KEOPS complex for 1 h.The mixture was applied to purification by Ni-NTA affinity chromatography and unbound proteins were eluted with lysis buffer supplemented with increasing concentrations of imidazole.The final eluted fractions containing KEOPS and potential interactors were denatured and reduced with 2 M guanidine hydrochloride and 10 mM TCEP at 56 • C for 30 min.The samples were subsequently alkylated using 40 mM iodoacetamide at room temperature in the dark, followed by overnight digestion using 2 μg trypsin.Tryptic peptides were acidified with 10% formic acid and then loaded on C-18 (3M) stage tips, desalted with 0.1% formic acid and eluted with buffer (80% acetonitrile, 0.1% formic acid).Samples were dried in a vacuum centrifuge.Dried peptides were dissolved in 0.1% formic acid and chromatographed using a C18 column (75 μm inner diameter, 15 cm length, 2 μm particles) on the EASY-nLC1000 UHPLC (ThermoFisher Scientific) coupled to Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific).Mass spectra were acquired in datadependent mode and MS measurements were constructed in the positive-ion mode.The m / z range was set to 350-1550, orbitrap resolution is 60 000.MS raw files were processed with the MaxQuant software (version 1.6.14)using standard settings with the additional options match between runs, iBAQ (intensity-based absolute quantification) selected.Other parameters were set up using the default values and the false discovery rate was set to 0.01 for both peptide and protein identification.Relative abundance of the identified interactors was measured by iBAQ values.
We first constructed an expression plasmid for A. thaliana YRDC using chemically synthesized DNA and purified recombinant YRDC ( Supplementary Figure S1 B) that was expressed bacterial cells.Liquid chromatography-mass spectrometry (LC-MS) analysis shows that YRDC is active in catalyzing the formation of threonylcarbamoyl-AMP (TC-AMP) in the presence of ATP, L -threonine and NaHCO 3 ( Supplementary Figure S1 B).
We constructed expression plasmids of A. thaliana KEOPS proteins using chemically synthesized DNAs and tried heterologous expression of KAE1-BUD32-PCC1-CGI121 (KEOPS), BUD32-CGI121 (BC), KAE1-PCC1 (KP), KAE1-BUD32 (KB), KAE1-BUD32-PCC1 (KBP) and CGI121 (C).We did not succeed in concomitantly expressing KAE1, BUD32, CGI121 and PCC1 using a polycistronic plasmid similar to that for S. cerevisiae ( Sc ) KEOPS ( 56 ).Alternatively, we assembled KEOPS by mixing KP and BC at a molar ratio of 1:2, followed by purification using size-exclusion chromatography (SEC) (Figure 1 A).The SEC and SDS-PAGE analysis of KEOPS complex, subcomplexes (KBP , KP , BC, KB) and CGI121 are shown in Supplementary Figure S1 C and Figure 1 B, respectively .Notably , we observed a persistent degradation KAE1 via the N-terminal end, which was confirmed by MS and western blot against 6His tag.SEC analysis shows that the four-subunit At KEOPS eluted out (at a calculated molecular weight of ∼200 kDa) earlier than the five-subunit Sc KEOPS (a monomeric complex with a molecular weight of 117.7 kDa) ( 56 ), indicating that At KEOPS exists as an eight-subunit dimer (K 2 B 2 C 2 P 2 ) as that of Mj KEOPS ( 44 ) ( Supplementary Figure S1 C).Compared SEC profiles suggest that either KAE1-PCC1 or KAE1-BUD32-PCC1 forms a dimer whereas BUD32-CGI121 or KAE1-BUD32 exists as a monomer ( Supplementary Figure S1 C).
We first tested the t 6 A-catalytic activity of At KEOPS using bulk Sc tRNAs that were isolated from S. cerevisiae sua5 cells.LC-MS analysis shows that Sc tRNAs were deficient in t 6 A but acquired t 6 A in the presence of At KEOPS and At YRDC (Figure 1 C).In addition, the catalytic activity of At KEOPS towards Sc tRNAs was comparable to that of Sc KEOPS (Figure 1 C).In summary, the active recombinant proteins expressed in E. coli cells demonstrate that folding and function of KEOPS do not require essential post-translational modifications for in vitro biosynthesis of tRNA t 6 A. We transcribed and purified 9 ANN-decoding tRNAs-tRNA Arg UCU , tRNA Arg CCU, tRNA Thr CGU , tRNA Ser GCU , tRNA Lys UUU , tRNA Ile AAU , tRNA Ile UAU , tRNA Met CAU and tRNA Asn GUUthat are encoded in A. thaliana nuclear genome (Figure 1 D).Electrophoretic mobility shift assay (EMSA) confirmed an interaction between At KEOPS and each of these in vitro transcribed (IVT) tRNAs (Figure 1 E).We then performed t 6 A assays using these IVT At tRNAs and analyzed t 6 A formation by LC-MS analysis ( Supplementary Figure S1 D).Normalized levels of t 6 A against A, U, C or G according to the nucleotide sequence showed great variations in t 6 A modification efficiencies of At KEOPS towards these IVT At tRNAs (Figure 1 F).At KEOPS exhibits strong activities on tRNA Arg CCU and tRNA Arg UCU , and gradually lowering activities on tRNA Thr CGU , tRNA Ile AAU , tRNA Lys UUU and tRNA Ser GCU .In comparison, tRNA Met CAU , tRNA Asn GUU and tRNA Ile UAU were not t 6 A-modified by At KEOPS.To exclude the possible problem in preparation of IVT tRNAs, we also deterimined different t 6 A-modification variations in these IVT At tRNAs by Sc KEOPS ( Supplementary Figure S1 E and  F).Our analysis suggests that the t 6 A modification frequencies are dependent on sequences of At tRNAs.In our case, At tRNA Ile UAU and At tRNA Met CAU lack a 36-UAA-38 motif (Figure 1 D), which is a determinant of t 6 A modification ( 55 ).We mutated 36-UGA-38 motif of At tRNA Ile UAU (Figure 1  variant-At tRNA Ile UAU -G37A.Indeed, At tRNA Ile UAU -G37A is t 6 A-modified by At KEOPS (Figure 1 F).As for the inactivity of At tRNA Asn GUU , we confirmed the overall folding by CD spectra analysis ( Supplementary Figure S1 D).Promoted by a recent discovery of a novel function of P. salinus TsaN in generating t 6 ATP / t 6 ADP using TC-AMP and ATP / ADP ( 47 ), we also analyzed the formation of t 6 ATP / t 6 ADP by KEOPS via detecting t 6 A-the dephosphorylated products of t 6 ATP / t 6 ADP.Our LC-MS analysis shows that At KEOPS is inactive in generating t 6 ATP / t 6 ADP in the presence of ATP and TC-AMP while P. salinus T saN 1-392 (T saD domain) catalyzes the formation of t 6 ATP / t 6 ADP in conjunction with At YRDC ( Supplementary Figure S1 B).
As At KEOPS possesses the highest activity on At tRNA Arg CCU , we set out to test the t 6 A-catalytic activities of At KEOPS subcomplexes in assays using IVT At tRNA Arg CCU as substrate (Figure 1 G and 1H).Our data demonstrates that KAE1-BUD32-PCC1 subcomplex is still capable of catalyzing the t 6 A formation of At tRNA Arg CCU , but the activity is decreased by around 50% compared that of KEOPS.KAE1-PCC1 subcomplex is no longer active in catalyzing the t 6 A modification of At tRNA Arg CCU (Figure 1 F).In addition, we show that At KEOPS still catalyzes t 6 Amodification of tRNA Arg CCU CCA in which the 3 CCA end was chopped (Figure 1 G and 1 H).In sum, our data show that BUD32 is strictly needed by At KEOPS to catalyze the biosynthesis of tRNA t 6 A while CGI121 is dispensable for in vitro tRNA t 6 A biosynthesis.In contrast, Mj Cgi121 directly binds tRNA via the 3 CCA end and plays a pivotal role in tRNA binding and t 6 A-catalytic function of Mj KEOPS ( 45 ).

Cryo-EM structure of A. thaliana KEOPS complex
We set out to determine the structure of At KEOPS-tRNA Arg CCU by single particle cryo-electron microscopy (cryo-EM).We were not able to isolate a preformed At KEOPS-tRNA Arg CCU complex by SEC (data not shown).Alternatively, a mixture of freshly purified At KEOPS and At tRNA Arg CCU (at a molar ratio of 1:2) was applied to cryo-EM grids for structural analysis.We optimized the vitrification and collected a dataset of 14 622 micrographs on a Titan Krios microscope operated at 300 keV.We analyzed the complete dataset by applying manual picking and topaz training as well as iterative rounds of particle picking.With 232 232 picks, we determined a map of At KEOPS at an overall resolution of 3.2-5.5Å ( Supplementary Figure S2 and Supplementary Table S3 ).We retrieved prediction models of At KEOPS subunits from AlphaFold Protein Structure Database ( 61 ) and generated fitting models for subcomplexes of KAE1-PCC1, KAE1-BUD32, BUD32-CGI121 and PCC1-PCC1 based on crystal structures of Mj Kae1-Pcc1 ( 44 ), Mj Kae1-Bud32 ( 3 ), Sc Bud32-Cgi121 ( 56 ) and P. furiosus ( Pf ) Pcc1-Pcc1 ( 44 ), respectively.The reconstruction shows six KEOPS subunits-KAE1, PCC1, PCC1, KAE1, BUD32 and CGI121, but no presence of a tRNA molecule (Figure 2 A).The dissociation of BUD32-CGI121 from KAE1-PCC1 in one KEOPS protomer was probably due to the structural damage at air-water interface during sample preparation.Imposing local refinement ( Supplementary Figure S2 B and C), we managed to determine almost a complete structure of At KEOPS complex with exception for the C-terminal tail of KAE1 (residues 341-353), N-terminal end (residues 1-13) and C-terminal tail (residues 222-226) of BUD32, and N-terminal end of PCC1 (residues 1-15).The map was blurred for two flexible loops (residues 36-48 and 173-179) of KAE1, which were modelled with references to crystal structures of its orthologs ( Supplementary Figure S3 A).We modelled a Fe 2+ ion in the binding sphere of H113-H117-D298 motif in KAE1 based on crystal structures of Kae1 / OSGEP proteins ( 3 , 18 , 52 ).The final model is composed of a complete At KEOPS protomer (PCC1-KAE1-BUD32-CGI121) and an incomplete protomer that only contains KAE1-PCC1 (Figure 2 A).Based on the dimeric state of At KEOPS complex in solution as determined by biochemical characterizations (Figure 1 B and Supplementary Figure S1 C), we aligned the structure of a complete KEOPS protomer to the structure of KAE1-PCC1 and completed a model of BUD32-CGI121 in relation to KAE1-PCC1 in the other protomer.In the reconstructed model of At KEOPS dimer, the two protomers interact via an interacting interface of PCC1 dimer and adopt a V-shaped architecture (Figure 2 B).

The structural uniformity and variation of KEOPSs
KEOPSs function as molecular machineries ( 31 ).Up to date, no atomic structures of a complete KEOPS complex are available except for a composite model of KEOPS from archaea ( 3 ) and crystal structures of KEOPS subcomplexes from yeast ( 56 ) and humans ( 18 ,57 ).We compared these KEOPS structures with At KEOPS to gain insights into functions.Overall, At KEOPS subunits manifest a substantial structural conservation to their orthologs from archaea, yeast and humans ( Supplementary Figure S3 ).RMSD values of structural alignment coupled sequence identities are summarized in Table 1 .Crystal structures of Hs LAGE3-OSGEP binary complex ( 18 ) and Hs PRPK-TPRKB binary complex ( 57 ) are perfectly superposed with structure of At KEOPS (Figure 2 C).Likewise, crystal structures of Sc Pcc1 ( 56 ) and Sc Bud32-Cgi121 binary complex ( 56 ) are satisfactorily aligned with structure of At KEOPS (Figure 2 C) except for Kae1 whose structure has not been determined.As for archaean KEOPS, crystal structures of Pf Pcc1-Mj Kae1 binary complex and Mj Bud32-Cgi121 binary complex are also satisfactorily superposed to PCC1-KAE1 and BUD32-CGI121 in the cryo-EM structure of At KEOPS, respectively ( Supplementary Figure S3 E).In sum, the overall arrangement and inter-subunit interfaces of KEOPS subunits are extremely conserved among KEOPSs.However, structural juxtaposition of At KEOPS and Mj KEOPS as a whole reveals a marked variation in the Clobes of BUD32 and Bud32 (Figure 2 D).In general, the C-lobe of At BUD32 is located farther away (at least 4 Å) than that of Mj Bud32 from the C-terminal domain of KAE1.Consequently, C-lobe makes fewer contacts with KAE1 (Figure 2 D).Superposition of At PCC1 dimer and Pf Pcc1 dimer exhibits conserved V-shaped architectures but varied conformations of At KEOPS dimer and Mj KEOPS dimer ( Supplementary Figure S3 F).
Unfortunately, tRNA Arg CCU was not resolved in the cryo-EM structure of At KEOPS.We made use of Mj KEOPS-tRNA model to gain insights into the binding of tRNA to At KEOPS.Structural juxtaposition of Mj KEOPS-tRNA to At KEOPS dimer generated a model for a dimer of At KEOPS-tRNA (Figure 2 E).According to this model, tRNA could be accommodated on an extended surface of the four subunits of At KEOPS.According to the docked model, anticodon stem loop of tRNA is sandwiched between KAE1 and PCC1; D stem loop protrudes between KAE1 and BUD32; 3 CCA end

KEOPS dimer is required to form a t 6 A-catalytic KEOPS-tRNA assembly
Based on high t 6 A-catalytic activity of At KEOPS on At tRNA Arg CCU , we chose to use 5 6-FAM (6-Carboxyfluorescein)-labelled tRNA Arg CCU (5 -6FAM-tRNA Arg CCU ) for interaction analysis.Our gel analysis shows a strong interaction between At KEOPS and 5 -6FAM-tRNA Arg CCU (Figure 3 A).In contrast, neither PCC1-KAE1 nor CGI121 is capable of binding 5 -6FAM-tRNA Arg CCU .In addition, our analysis revealed a relatively weak interaction between 5 -6FAM-tRNA Arg CCU and PCC1-KAE1-BUD32 or BUD32-CGI121 (Figure 3 A).We further measured the binding affinities with microscale thermophoresis (MST) (Figure 3 B) and determined equilibrium constants ( K d ) for the interactions between 5 -6FAM-tRNA Arg CCU and At KEOPS proteins (Table 2 ).We determined a K d value of 18.08 μM for the interaction between 5 -6FAM-tRNA Arg CCU and At KEOPS.In comparison, K d values for PCC1-KAE1-BUD32 and BUD32-CGI121 towards 5 -6FAM-tRNA Arg CCU are 37.99 μM and 61.58 μM, respectively.Consistent with EMSA analysis, no binding events were observed with 5 -6FAM-tRNA Arg CCU and PCC1-KAE1 or CGI121 by MST measurements (Figure 3 B).These binding data demonstrate that CGI121 does not bind tRNA but promotes the binding of tRNA to KEOPS whereas BUD32 is strictly needed for binding of tRNA to At KEOPS.
Our interaction analysis demonstrates a central role of BUD32 in tRNA binding.However, the model of At KEOPS-tRNA does not support a direct contact between BUD32 and tRNA (Figures 2 E and 3 C).In contrast, the model of Mj KEOPS-tRNA coupled mutational validations demonstrated that the C-lobe of Mj Bud32 interacts with D stem loop and amino acid acceptor stem of Mj tRNA Lys UUU ( 45 ).We presumed that the C-lobe of At BUD32 is also primarily involved in binding of tRNA.We manually adjusted the position of tRNA by means of aligning the C-lobe of Mj Bud32 in rigid complex with Mj tRNA Lys UUU to the C-lobe of At BUD32, leading to an adjusted model of At KEOPS-tRNA (Figure 3 C).In this model, tRNA was rotated by 15 • and shifted by 16 Å towards the C-lobe of At BUD32, giving better geometrical complementarity and electrostatic interactions (Figure 3 D).Such a manual rotation of Mj tRNA Lys UUU from crystal structure of Mj Cgi121-tRNA Lys UUU was also performed to fit the geometry of Mj KEOPS ( 45 ).It further suggests that assembly of KEOPS-tRNA complex might involve large conformational changes in KEOPS subunits and tRNA.
We set out to find out whether At KEOPS dimer is needed for tRNA t 6 A biosynthesis.To disrupt the PCC1 dimer, we simultaneously mutated the Arg70, Arg73 and Ala74 of PCC1 (Figure 3 E) and generated an At KEOPS variant-PCC1 R70D / R73D / A74E -KAE1-BUD32-CGI121 (P R70D / R73D / A74E KBC) ( Supplementary Figure S4 A  and B).SEC analysis demonstrates that P R70D / R73D / A74E KBC exists exclusively as a monomer with an elution volume roughly overlapping with that of the five-subunit Sc KEOPS (Figure 3 E).Hereafter, P R70D / R73D / A74E KBC is dubbed as At KEOPS monomer and the dimer refers to wild-type At KEOPS.The interaction between At KEOPS monomer and 5 -6FAM-tRNA Arg CCU becomes apparently weaker than that of At KEOPS dimer and 5 -6FAM-tRNA Arg CCU (Figure 3 F).MST measurements determined a K d value of 74.37 μM for the interaction between At KEOPS monomer and 5 -6FAM-tRNA Arg CCU (Figure 3 B).Our LC-MS analysis shows that At KEOPS monomer is no longer active in catalyzing t 6 A biosynthesis (Figure 3 G).To find out whether the loss of t 6 A-ctalytic activity is due to disruption of dimer but not the loss of Arg70, Arg73 or Ala74 (as functional sites), we mutated Arg73 of PCC1 and generated an At KEOPS variant-PCC1 R73D -KAE1-BUD32-CGI121 (P R73D KBC).SEC analysis shows that P R73D KBC elutes out between At KEOPS dimer and monomer ( Supplementary Figure S4 A  and B), suggesting that P R73D KBC exists as mixture of dimer and monomer.Another explanation of the SEC profile is that the R73D mutation possibly induces large conformational changes in the dimeric architecture.EMSA analysis and MST measurements exhibit a significantly weaker interaction ( K d = 352.7 μM) between P R73D KBC and 5 -6FAM-tRNA Arg CCU ( Supplementary Figure S4 C and D).However, P R73D KBC still sustains around 25% t 6 A-catalytic activity of At KEOPS dimer ( Supplementary Figure S4 E).In sum, our structural analysis and functional validation show that At KEOPS dimer is needed to form a t 6 A-catalytic KEOPS-tRNA assembly.
Characterization KAE1 reveals regulatory sites related to t 6 A-catalytic activity of KEOPS KAE1 adopts a typical two-subdomain fold that is conserved in all TsaD / Kae1 / Qri7 / OSGEP structures ( Supplementary Figure S3 A).The t 6 A-catalytic site is located between the two subdomains and a divalent metal ion (Fe 2+ / Zn 2+ ) is essential for TC-AMP binding and t 6 A catalysis ( 32 , 41 , 54 ).Based on the model of KEOPS-tRNA complex (Figure 3 C) and loss-of-function mutations in Hs OSGEP, we chose to characterize the functional sites of At KAE1 (Figure 4 B): (i) an extremely conserved H-H-D motif (H117 and D298) that coordinates the metal ion Fe 2+ ( 32 ); (ii) I17, K202 and R284 that are equivalent to missense mutations (I14F, K198R, R280C) of Hs OSGEP in GAMOS patients ( 16); (iii) non-conserved A231 and Y305 in the vicinity of the catalytic site of KAE1.We generated these mutations and purified corresponding KEOPS variants: K H117A BCP, K D298R BCP , K I17F BCP , K K202R BCP , K R284C BCP , K A231G BCP and K Y305A BCP ( Supplementary Figure S5 A and B).Our gel interaction analysis shows that 5 -6FAM-tRNA Arg CCU is almost completely bound to these KEOPS variants when mixed at a molar ratio of 1:5 ( Supplementary Figure S5 C).However, quantitative analysis by MST reveals that the interactions between 5 -6FAM-tRNA Arg CCU and these KEOPS variants are weaker than that between 5 -6FAM-tRNA Arg CCU and wild-type KEOPS (Figure 4 C), as indicated by increased K d values for these KEOPS variants (Table 2 ).Notably, I17F severely interferes with the binding of tRNA to KEOPS.We performed assays on t 6 A catalysis of these KEOPS variants ( Supplementary Figure S5 E and F) and show different effects of these mutations on t 6 A-catalytic activity of KEOPS (Figure 4 D): 1) K202R mutation confers less effect on t 6 A-catalytic activity of KEOPS; 2) either I17F mutation or A231G mutation results in half of the t 6 A-catalytic activity; 4) H117A, D298R or R284C leads to dead t 6 A-catalytic activity; 5) Y305A mutation also causes a dead t 6 A-catalytic activity as only trace amount of t 6 A was detected.In light of the structural model (Figure 4 B), our analysis of the interaction and t 6 A-catalysis of these mutations of KAE1 confirms an essential role of H117 and D298 in t 6 A catalysis.Interestingly, no degradation of KAE1 was observed in K H117A BCP and K D298R BCP ( Supplementary Figure S5 B).Our analysis suggests that I17, Y305 and A231 might modulate the configuration of t 6 A-catalytic center whereas K202 might participate in tRNA binding and R284 might modulate the function of BUD32.
The precise binding of anticodon stem loop of tRNA to catalytic sites of TsaD / Kae1 / Qri7 proteins still remains undetermined and poses an obstacle in mechanistic understanding of KEOPS.Our model of KEOPS-tRNA shows that the connecting loop (residues 36-48) between β1 and α1 of KAE1 adopts a conformation that creates steric clashes with incoming anticodon stem loop of tRNA (Figure 3 C).
Sequence and structural alignment show that these connecting loops in eukaryotic Kae1 / KAE1 / OSGEP proteins are highly conserved (Figure 4 A) and conformationally divergent ( Supplementary Figure S3 A).However, the functions of these loops of TsaD / Kae1 / Qri7 family proteins remain unexplored.We generated two At KEOPS variants with multiple mutations in the loop connecting β1 and α1 of KAE1-K Y36R / I37K / T38R / P39R / P40R / G41W / H42D BCP (K mutant-1 BCP) and K G43E / F44R / L45K / P46R / R47D / E48K BCP (K mutant-2 BCP).We substituted non-charged residues with positively-charged residues of arginine and lysine in these two mutants.In K mutant-2 BCP, we additionally substituted positively-charged Arg47 with Asp and negatively-charged Glu48 with Lys, respectively.Our purification and SEC analysis demonstrate that these multiple mutations do not affect solubility of KAE1 and overall structure of KEOPS ( Supplementary Figure S5 A and B).Our gel analysis shows that both K mutant-1 BCP and K mutant-2 BCP are capable of binding 5 -6FAM-tRNA Arg CCU ( Supplementary Figure S5 D).Notably, MST measurements demonstrate an enhanced interaction between K mutant-1 BCP and 5 -6FAM-tRNA Arg CCU ( K d = 5.27 μM) but no interaction between K mutant-2 BCP and 5 -6FAM-tRNA Arg CCU (Figure 4 C).Both K mutant-1 BCP and K mutant-2 BCP lose the t 6 A-catalytic activity ( Supplementary Figure S5 E, Figure 4 D), suggesting that the connecting loop between β1 and α1 of KAE1 is essentially required for tRNA t 6 A biosynthesis by KEOPS.In particular, our model of KEOPS-tRNA and interaction analysis suggest that the first half of the loop (residues 36-42) might participate in tRNA binding (Figure 4 B), as K mutant-1 BCP has acquired increased affinity towards tRNA (Figure 4 C).

KEOPS is modulated by BUD32 via the C-terminal tail and ATP to ADP hydrolysis
Our model of KEOPS-tRNA coupled interaction analysis features an essential role of BUD32 in direct binding of tRNA to KEOPS (Figure 3 D, Figure 5 A).The model suggests that the 3 amino acid acceptor arm (nucleobases 66-71) of tRNA is located in close proximity to α1 and α5 of BUD32 (Figure 5 A).To confirm the participation of α1 and α5 of BUD32 in tRNA binding, we generated KEOPS variants bearing mutations in α1 (I47K, K51E, K55E and N58R) and in the connecting loop of β8 and α5 (T162R, S163R and L165K) of BUD32.We purified these BUD32-mutated KEOPS variants-KB I47K CP, KB K51E CP, B K55E CP, KB N58R CP, KB T162R CP, KB S163R CP and KB L165K CP ( Supplementary Figure S6 A and B).We first measured the interactions between 5 -6FAM-tRNA Arg CCU and KEOPS variants by EMSA and MST.EMSA analysis shows that K55E mutation severely affects the interaction between 5 -6FAM-tRNA Arg CCU and KEOPS; K51E, T162R or S163R mutation confers a milder effect; I47K, N58R or L165K mutation exerts no effect ( Supplementary Figure S6 C).However, quantitative analysis by MST demonstrates that I47K, K55E, S163R, L165K or K51E mutation leads to weaker interaction between 5 -6FAM-tRNA Arg CCU and KEOPS while N58R or T162R mutation slightly promotes the binding of 5 -6FAM-tRNA Arg CCU to KEOPS (Figure 5 B, Table 2 ).It seems that substitution of Lys55 by negatively-charged Glu leads to markedly weak binding affinity of KEOPS towards 5 -6FAM-tRNA Arg CCU whereas substitution of Asn58 and Thr162 by positively-charged Arg enhances the binding affinities, suggesting that Lys55, Asn58 and Thr162 participate in tRNA binding.We then measured the t 6 A-catalytic activities of these KEOPS variants ( Supplementary Figure S6 D, Figure 5 C) and demonstrate that K55E mutation substantially compromises t 6 A-cataalytic activity of KEOPS; K51E or T162R mutation mildly affects the t 6 A-catalytic activity; I47K, N58R, S163R or L165K mutation confers negligible effect (Figure 5 C).Nonetheless, K55E mutation causes a drop in both binding affinity and t 6 A-catalytic activity, strongly documenting a role for Lys55 of BUD32 in tRNA binding.
Our cryo-EM structure of At KEOPS reveals distinct conformation of the C-lobe of BUD32 in relation to KAE1 compared to that of Mj KEOPS.Strikingly, the C-terminal tail of BUD32 adopts a unique conformation (Figure 3 C).In both structures, the C-terminal tails of BUD32 / Bud32 protrude towards the catalytic site of KAE1 / Kae1.However, the structure of the tail sequence ( 220 RKRTMIG 226 ) was not observed in our structure (Figure 5 D) nor in structures of BUD32 orthologs ( 3 , 56 , 57 ).Sequence alignment reveals that the tail sequences are highly conserved in eukaryotic BUD32 / PRPK / Bud32 proteins but less conserved in archaean Bud32, which lacks a MxG motif (Figure 5 E).The C-terminal tails of BUD32 / PRPK / Bud32 proteins contain a positively-charged RKR motif (Figure 5 E).To analyze the function of the C-terminal tail of BUD32, we generated a series of KEOPS variants, including a variant lacking 220 RKRTMIG 226 (KB R220stop CP) and eight variants with single mutation-KB R220A CP, KB K221A CP, KB R222A CP , KB T223A CP , KB M224A CP , KB I225A CP , KB G226A CP and KB G226R CP (Figure 5 D).We purified good quality proteins of these KEOPS variants ( Supplementary Figure S7 A  and B).Our EMSA analysis shows that the binding of 5 -6FAM-tRNA Arg CCU to KEOPS is not essentially affected or disrupted by deletion of 220 RKRTMIG 226 or single mutation in it ( Supplementary Figure S7 C).MST measurement demonstrates that the interaction between 5 -6FAM-tRNA Arg CCU and KB R220A CP or KB R220stop CP was weaker than wild-type KEOPS (Figure 5 F).The K d values for KEOPS, KB R220stop CP and KB R220A CP are 18.08 μM, 148.39 μM and 88.20 μM, respectively.We performed tRNA t 6 A assay using IVT At tRNA Arg CCU and these variants ( Supplementary Figure S7 D), and detected no t 6 A with KB R220stop CP and only trace amount of t 6 A with KB R220A CP, respectively (Figure 5 G).In contrast, we detected large amount of t 6 A that is comparable to wild-type KEOPS in assays using KB K221A CP, KB M224A CP , KB I225A CP , KB G226A CP or KB G226R CP .In parallel, we detected decreased levels of t 6 A in assays using KB R222A CP or KB T223A CP.These data demonstrate that Arg220 of BUD32 plays an essential role in regulating the t 6 A-catalytic activity of KEOPS.
Previous studies demonstrated that Bud32 / PRPK regulates the function of KEOPS via an ATP to ADP hydrolysis-based mechanism ( 20 ,45 ).We first measured the ATPase activity of At KEOPS complex and subcomplexes using an NADHcoupled ATPase assay.We determined an efficient conversion of ATP to ADP in the presence of KAE1-BUD32, PCC1-KAE1-BUD32 or KEOPS, but neither KAE1-PCC1 nor BUD32-CGI121 ( Supplementary Figure S6 E, Figure 5 H).Comparison of the ATP hydrolysis rates (reaction velocity in steady state) demonstrate that BUD32 catalyzes the ATP hydrolysis and KAE1 stimulates the ATPase activity of BUD32, which is further potentiated by CGI121 (Figure 5 H).Structural juxtaposition of At BUD32 and Hs PRPK in complex with ATP and Mg 2+ projects ATP and two Mg 2+ ions in the catalytic site of At BUD32 (Figure 5 D), which reveals that the conserved D137 and D156 might participate in coordination of ATP and Mg 2+ (Figure 5 D).We mutated D137 and D156 of BUD32 and generated two KEOPS variants-KB D137A CP and KB D156A CP ( Supplementary Figure S6 A and B).We show that KB D137A CP and KB D156A CP is no longer active in hydrolyzing ATP to ADP (Figure 5 H).Meanwhile, KB R220stop CP exhibits an ATPase activity that is comparable to the KEOPS (Figure 5 H), suggesting that the C-terminal tail of BUD32 does not directly participate in A TP hydrolysis.W e further determined that IVT At tRNA Arg CCU markedly potentiates the ATP hydrolysis by KEOPS (Figure 5 H).We measured catalytic kinetics of ATP hydrolysis by 2 μM KEOPS (Figure 5 I): In parallel, we determined catalytic kinetics of 2 μM KEOPS in the presence of 10 μM IVT At tRNA Arg CCU :

Proteomic analysis of KEOPS-interacting proteins
Recently, Daugeron et al. identified no orthologs of Gon7 / Pcc2 in Arabidopsis thaliana using advanced comparative genomic analysis ( 46 ).Our in vitro assay also confirmed a t 6 A-catalytic activity of At KEOPS on IVT tRNAs.Nonetheless, we performed proteomic analysis of KEOPS-interacting proteins using LC-MS / MS.We simply incubated purified KEOPS with total soluble proteins isolated from Arabidopsis thaliana seedlings and purified potential interactors of 6His tagged KEOPS by Ni-NTA chromatography.Unbound proteins were removed by repeated washing using lysis buffer until no proteins appeared in the wash fraction prior to final elution of KEOPS.SDS-PAGE analysis showed that the final elution sample contained four subunits of KEOPS and very weak bands of proteins (data not shown).
The final elution sample were digested into tryptic peptides and applied to LC-MS / MS for proteomic analysis.In total, 671 proteins were identified in the MS samples and listed in order according to the intensity-based absolute quantification (iBAQ) values ( Supplementary Table S4 ).The list contains 664 characterized proteins with functions implicated in a wide range of cellular processes and 7 uncharacterized proteins (Uniprot IDs: Q9FKA5, A0A1P8B3M2, Q9C6U3, O64818, Q9FZG2, Q93W28 and F4J0L7).Notably, Q9FZG2 has a molecular size of 10.67 kDa, which is close to that of GON7 / Gon7.Interestingly, AlphaFold prediction structure of Q9FZG2 (entry: Q9FZG2, www.alphafold.ebi.ac.uk ) shows that Q9FZG2 is an intrinsically disordered protein with a structured core comprising two antiparallel β-stands connected to an α-helix.

Discussion
Evolutionarily conserved KEOPS catalyzes the transfer of TCmoiety from TC-AMP to N6 atom of adenine at position 37 of tRNA, leading to tRNA t 6 A. Up to date, characterizations of KEOPSs ( Methanococcus jannasc hii , Sacc haromyces cerevisiae and Homo sapiens ) revealed structures, overall arrangement and functions of KEOPS subunits ( 31 ).However, the structure of a complete KEOPS and molecular workings of KEOPS still remain unresolved.In the present study, we determined a cryo-EM structure of KEOPS complex from Arabidopsis thaliana and performed structure-function relationship analysis of A. thaliana KEOPS.The data analysis and findings are discussed below.We successfully purified recombinant KEOPS proteins and reconstituted a stable KEOPS complex that is composed of KAE1, BUD32, CGI121 and PCC1.We show that the foursubunit A. thaliana KEOPS is active in catalyzing tRNA t 6 A biosynthesis in conjunction with recombinant A. thaliana YRDC (Figure 1 C).Though no ortholog of GON7 / Gon7 in Arabidopsis thaliana has been identified using bioinformatic analysis ( 46 ), we identified a large number of potential KEOPS interactors using pull-down assay and LC-MS / MS proteomic analysis ( Supplementary Table S4 ).Of note, the uncharacterized Q9FZG2 (At1g47820) and O64818 (At2g23090) possess GON7 / Gon7 features-comparable molecular sizes and partial intrinsically disordered structures.The structures and functions of Q9FZG2 and O64818 remain to be experimentally determined.Moreover, the presence of other proteins in MS sample eluted with KEOPS suggests that KEOPS or subunits might interact weakly or transiently with identified proteins.However, such a hypothesis remains to be experimentally validated in future.
A number of studies demonstrated large variations in t 6 A modification frequencies of KEOPS towards different tRNA substrates ( 20 , , 45 , 55 ).Our in vitro assay shows that the t 6 A-catalytic activities of KEOPS towards IVT tRNAs are different (Figure 1 F).KEOPS exhibits very low t 6 A-catalytic activity on tRNA Ser GCU and no activity on tRNA Asn GUU , tRNA Met CAU and tRNA Ile UAU , of which the latter two substrates lack a 36-UAA-38 motif.Introduction of a 36-UAA-38 motif into tRNA Ile UAU restores the t 6 A modification by KEOPS (Figure 1 F).As per the inactivity of tRNA Asn GUU and low activity tRNA Ser GCU , other native modifications might be needed to stabilize the active conformations of these two tR-NAs for more efficient t 6 A modification.The other explanation is that the in vitro folding of IVT tRNAs does not adopt a t 6 A-productive conformation, though our SEC, CD and interaction analysis shows that these IVT tRNAs adopt comparable overall 3D structures.
We determined a K d value of 18 μM for the binding of KEOPS with IVT tRNA Arg CCU and attempted to determine a cryo-EM structure of KEOPS-tRNA Arg CCU .Unfortunately, it turned out that tRNA was absent in the cryo-EM structure.Moreover, BUD32-CGI121 of one protomer of the KEOPS dimer was not observed in the structure (Figure 2 A).We presume that the interaction between KEOPS and tRNA Arg CCU was too weak to form a stable complex in the EM sample, as we were not able to purify a KEOPS-tRNA Arg CCU by SEC.In another scenario, a fraction of tRNA Arg CCU bound to BUD32-CGI121 that was dissociated from KEOPS in the EM sample, as the K d for the interaction between BUD32-CGI12 and tRNA Arg CCU is 62 μM.Our cryo-EM structure of At KEOPS complex reveals a conserved overall arrangement of KEOPS subunits ( 31 ).The model of Mj KEOPS-tRNA provides a good framework for mechanistic understanding of KEOPS ( 45 ).It shows that tRNA binds to an extended surface of KEOPS and makes contacts with the four subunits ( 31 ,45 ).We generated a rigid model for A. thaliana KEOPS-tRNA by means of structural juxtaposition of At KEOPS and Mj KEOPS-tRNA (Figure 2 E).We further manually refined the geometric and electrostatic fitting of tRNA to KEOPS with reference to conformation of the C-lobes of Mj Bud32 / At BUD32, which is directly involved in tRNA binding ( 45 ).The resulting model of KEOPS-tRNA reveals a good geometric complementarity and electrostatic interaction (Figure 3 D).The model features a central role of BUD32 in tRNA binding.Our mutational analysis of BUD32 supports a direct participation of α1, on which the K55E mutation leads to markedly weak interaction between KEOPS and tRNA Arg CCU (Figure 5 B) and decreased t 6 A-catalytic activity of KEOPS (Figure 5 B).In addition, a number of other mutations in the connecting loop of β8 and α5 of BUD32 also interferes with the interaction between KEOPS and tRNA Arg CCU .Moreover, we show that the loop connecting β1 and α1 of KAE1 participates in binding of tRNA to KEOPS (Figure 4 B).We determined that CGI121 or PCC1-KAE1 is not capable of binding tRNA but BUD32-CGI121 and KAE1-BUD32-PCC1 can independently bind tRNA (Figure 3 A and B).Subtraction of CGI121 from KEOPS or deletion of 3 CCA end of tRNA Arg CCU only reduces the t 6 A-catalytic activity of KEOPS (Figure 1 H).In contrast, Mj Cgi121 alone or Mj Bud32-Cgi121 is capable of binding tRNA but Mj Kae1-Bud32-Pcc1 does not bind tR-NAs ( 45 ).Pa Pcc1-Kae1 is a binding core for tRNAs and Pa Cgi121 alone does not bind tRNAs ( 20 ).The functional differences among different KEOPS proteins imply specific mechanisms underscoring the molecular interaction and regulation of KEOPS-tRNA assembly.However, mutational analysis of the interactions and t 6 A-catalytic activities justifies at least the binding orientation of tRNA to KEOPS in our model of At KEOPS-tRNA.
Our cryo-EM structure of A. thaliana KEOPS revealed a dimeric structure of KEOPS, which is consistent with the dimeric state of At KEOPS as determined by SEC.A tKEOPS dimer is mediated by homodimerization of PCC1 in a similar manner as for archaean KEOPS ( Supplementary Figure S3 E) ( 44 ).We generated a four-subunit KEOPS monomer (P R70D / R73D / A74E KBC) via disrupting the dimerization of PCC1.KEOPS monomer is capable of binding tRNA (Figure 3 F).However, the binding affinity for KEOPS monomer and tRNA Arg CCU ( K d = 74 μM) is close to that for BUD32-CGI121 and tRNA Arg CCU ( K d = 62 μM), which are greatly lower than the binding affinity of wild-type KEOPS dimer towards tRNA Arg CCU (Table 2 ).Nonetheless, KEOPS monomer is no longer active in biosynthesizing tRNA t 6 A (Figure 3 G).Our model of At KEOPS-tRNA shows that one KEOPS protomer could binds one tRNA, whose anticodon stem loop potentially makes contacts with PCC1-KAE1 from the other protomer (Figure 2 E, Figure 3 D).Therefore, we presume that correct binding of tRNA to one KEOPS protomer is facilitated by the other protomer .However , we cannot exclude the possibility that KEOPS dimer binds only one molecule of tRNA.In this case, binding of tRNA to one protomer precludes the binding of second tRNA to the other protomer.Such a similar mechanism is adopted by mitochondrial Qri7 dimer ( 34 ) and bacterial T saD2-T saB2 tetramer ( 38 ).Unfortunately, we could not determine the stoichiometry and number of binding sites by MST and EMSA measurements.In sum, our data shows that At KEOPS dimer is required to form a t 6 A-catalytic KEOPS-tRNA assembly.
ATP to ADP hydrolysis by BUD32 is involved in the turnover of t 6 A-catalysis by KEOPS.Such a mechanism is also adopted by bacterial TsaDBE complex ( 38 ,41 ).The ATPase catalytic site is sandwiched between the N-lobe and C-lobe of Bud32 / PRPK ( 56 ,57 ).We show that ATP to ADP hydrolysis by BUD32 is stimulated by KAE1 and further strongly potentiated by tRNA Arg CCU (Figure 5 H).Deletion of C-terminal tail or abrogation of ATPase activity of At BUD32 does not interfere with binding of tRNA to At KEOPS but leads to dead t 6 A-catalytic activity.But how the C-terminal tail and ATPase activity of BUD32 affect the t 6 A activity of KEOPS remains a long-standing question.Here we determined that Arg220 at the C-terminal tail of BUD32 is critical for the t 6 A-catalytic activity of Kae1.Our structure shows a distance of 25 Å between Arg220 of BUD32 and t 6 A-catalytic site of KAE1.Therefore, it's unlikely that Arg220 of BUD32 reaches into the t 6 A-catalytic site of KAE1 and modulates the TC-transfer reaction.We presume that Arg220 or the positively charged C-terminal tail of At BUD32 might play a pivotal role in stabilizing anticodon stem loop of tRNA.In structure of At KEOPS, the closest distance between α7 of KAE1 and ATPase catalytic site of BUD32 is less than 8 Å.We hypothesize that α7 of KAE1 interacts with α1 and α5 of BUD32 and stimulates the ATPase activity of BUD32.In turn, the hydrolysis of ATP to ADP drives the relocation of α7 of KAE1.By doing so, the two lobes of BUD32 undergo substantial conformational changes between the ATP-bound state and ADP-bound state.Movement of the C-terminal tail along with the relocation of the C-lobe of BUD32 might participate in binding and release of tRNA.Therefore, the C-terminal tail of BUD32 offers an opportunity to couple ATP hydrolysis with correct binding of tRNA to KEOPS, conferring an allosteric regulation of t 6 Acatalytic cycle of KEOPS.

Conclusion
We have determined a conserved role of A. thaliana KEOPS in tRNA t 6 A biosynthesis.Our structure-function relationship analysis of KEOPS-tRNA assembly suggests that the four-subunit KEOPS dimerize via PCC1 in support of a correct binding of tRNA to KEOPS, which requires the essential contribution of BUD32 and is further promoted by CGI121.BUD32 seems to be key regulator of KEOPS.The C-terminal tail of BUD32 functions as a 'trigger' to modulate the t 6 Acatalytic activity of KAE1.KAE1 stimulates the ATPase activity of BUD32 in exchange for turnover of the t 6 A-catalysis, which is driven by ATP to ADP hydrolysis by BUD32.Still, there are several fundamental questions to be addressed in order to mechanistically understand the inner workings and cellular roles of KEOPS machineries.In particular, the specific recognition of ANN-decoding tRNAs by KEOPSs and the chemistry of t 6 A catalysis by Kae1 / KAE1 / OSGEP await to be fully elucidated.A high-resolution structure of a complete KEOPS-tRNA complex is undoubtedly desirable to solve these mysteries.

Figure 1 .
Figure 1.In vitro enzymatic reconstitution of tRNA t 6 A biosynthesis using recombinant YRDC and KEOPS proteins of Arabidopsis thaliana .( A ) Siz e-e x clusion chromatograph y (SEC) profile (HiL oad 16 / 600 Superde x 200, GE Healthcare) of A. thaliana KEOPS and BUD32-CGI121.KEOPS w as reconstituted using purified KP (KAE1-PCC1) and BC (BUD32-CGI1 21), whic h w ere mix ed at a molar ratio of 1:2.( B ) SDS-PAGE analysis of KEOPS, KBP (KAE1-BUD32-PCC1), KP, BC, CGI121 and KB (KAE1-BUD32).* indicates KAE1 degradation (confirmed by LC-MS / MS). ( C ) LC-MS analysis of t 6 A formation in assay that contained 4 mM L -threonine, 20 mM NaHCO 3 , 2 mM ATP, 20 μM bulk Sc tRNAs (isolated from sua5 strain), 2 μM At YRDC and 2 μM At KEOPS or Sc KEOPS.( D ) Nucleotide sequences of A. thaliana tRNAs that were in vitro transcribed (IVT) and used for tRNA t 6 A assays.Amino acid acceptor arm, D stem loop, anticodon stem loop and T ψ C stem loop of tRNAs are highlighted in pink, green, blue and gray, respectively.Anticodons are shown in red.( E ) Native gel analysis of the interaction between 10 μM At KEOPS and 20 μM IVT At tRNAs (tRNA Arg CCU , tRNA Arg UCU , tRNA Thr CGU , tRNA Ile AAU , tRNA Lys UUU , tRNA Ser GCU , tRNA Met CAU , tRNA Asn GUU , tRNA Ile UAU and tRNA Ile UAU -G37A).( F ) Quantified t 6 A modification efficiencies of At KEOPS to w ards these IVT At tRNAs.Percentage of t 6 A was normalized according to tRNA sequence.( G ) LC-MS analysis of tRNA t 6 A f ormation b y 2 μM At YRDC and At KEOPS, At KBP or At KP using 20 μM IVT At tRNA Arg CCU or At tRNA Arg CCU -CCA as substrate.( H ) Quantified t 6 A modification efficiencies of At KEOPS, At KBP and At KP to w ards At tRNA Arg CCU or At tRNA Arg CCU -CCA (G).

Figure 5 .
Figure 5. Functional modulation of A. thaliana KEOPS by BUD32.( A ) The model of A. thaliana KEOPS-tRNA shows residues of BUD32 (colored in blue) in proximity to amino acid acceptor arm of tRNA.( B ) Microscale thermophoresis (MST) measurements of the interactions between 50 nM 5 -6FAM-tRNA Arg CCU and KEOPS variants bearing mutations in BUD32.( C ) Comparison of the t 6 A modification efficiencies of IVT At tRNA Arg CCU by KEOPS variants bearing mutations in BUD32.Error bars represent standard deviations for triplicate measurements.( D ) The interacting interface of KAE1 (colored in red) and BUD32 (colored in blue) in the model of A. thaliana KEOPS-tRNA.ATP and Mg 2+ ions were projected in the ATPase-catalytic site of BUD32 according to the crystal str uct ures of H. sapiens PRPK (PDB: 6WQX) and S. cerevisiae Bud32 (PDB: 4WW9).The C-terminal tail ( 222 RTMIG 226 ) as represented in dashed cylinder was not observed in cryo-EM str uct ure of A. thaliana KEOPS complex.tRNA A37 and Fe 2+ ion indicate catalytic center of KAE1.( E ) Local sequence alignment of the C-terminal tails of A. thaliana ( At ) BUD32, H. sapiens ( Hs ) PRPK, S. cerevisiae ( Sc ) Bud32 and M. jannaschii ( Mj ) Bud32. ( F ) MST measurements of the interactions between 50 nM 5 -6FAM-tRNA Arg CCU and At KEOPS, At KB R220stop CP or KB R220A CP.Error bars represent standard deviations for triplicate measurements.( G ) Comparison of the t 6 A modification efficiencies of A. thaliana KEOPS, KB R220stop CP or KB R220A CP to w ards IVT At tRNA Arg CCU .( H ) Hy droly sis rate of 2 mM ATP to ADP b y 5 μM A. thaliana KEOPS comple x, subcomple x es or v ariants in the presence or absence of 10 μM IVT At tRNA Arg CCU .( I ) Kinetic plots of h y droly sis of ATP to ADP by 2 μM A. thaliana KEOPS, K D298R P or KB D137A CP in the presence or absence of 10 μM IVT At tRNA Arg CCU .( J ) Comparison of the t 6 A modification efficiencies of A. thaliana KEOPS, KB D137A CP or KB D156A CP to w ards IVT At tRNA Arg CCU .

Table 1 .
Statistics of the str uct ural alignment coupled sequence identities for KEOPS subunits from A. thaliana , H. sapiens , S. cerevisiae and archaean species ( M. jannaschii, P. ab y ssi, T. acidophilum and P. furiosus ) The kinetic parameters indicate that tRNA potentiates hydrolysis of ATP to ADP by KEOPS but slightly reduces the binding affinity of ATP to KEOPS.Thus, it suggests that binding of tRNA to KEOPS directly induces a conformational change in the catalytic site of BUD32.We further determined that either KB D137A CP or KB D156A CP still sustains a very low level of t 6 Acatalytic activity ( Supplementary FigureS6D and Figure5 J), suggesting that deprivation of the ATPase activity of BUD32 severely interferes with turnover of t 6 A-catalysis by KEOPS.